分散液液微萃取

Dispersive by microvolume Saadat Rastegarzadeh Department of Chemistry,detection.Analysis of fungicide thiram in environmental and agricultural samples was performed.a r t i c l e i n f o Article history:Received 11February 2013Received in revised form 7May 2013Accepted 8May 2013Available online 23May 2013Keywords:ThiramDispersive liquid–liquid microextraction Microvolume spectrophotometrya b s t r a c tA novel and simple method for the sensitive determination of trace amounts of fungicide thiram is devel-oped by combination of dispersive liquid–liquid microextraction (DLLME)and microvolume UV–vis spectrophotometry.The method is based on the conversion of thiram to a yellow product in the presence of ethanolic potassium hydroxide and copper sulfate,and its extraction into CCL 4using DLLME technique.In this method the ethanol existing in ethanolic KOH plays as disperser solvent and a cloudy solution is formed by injection of only CCl 4as extractant solvent into sample solution.Under the optimum condi-tions,the calibration graph was linear over the range of 25–1000ng mL À1of thiram with limit of detec-tion of 11.5ng mL À1.The relative standard deviation (RSD)for 100and 500ng mL À1of thiram was 2.7and 1.1%(n =8),respectively.The proposed method was successfully applied to determination of thiram in water and plant seed samples.Ó2013Elsevier B.V.All rights reserved.IntroductionDithiocarbamates (DTCs)are a group of organosulfur com-pounds that have been extensively used as pesticides in agriculture and horticulture over several decades [1,2].DTCs can be catego-rized into three subclasses depending upon their carbon skeleton including dimethyldithiocarbamates (DMDs),ethylenebis (dithio-carbamates)(EBDs),and propylenebis (dithiocarbamates)(PBDs).Among these,thiram (tetramethyl thiuram disulfide)(Fig.1)is a well-known dithiocarbamate fungicide that has been in commer-cial use since 1925[3].Thiram is widely used to prevent crop dam-age in the field and to protect harvested crops from deterioration in storage or transport and is also used as a seed protectant to control anumber of fungal diseases [4].Therefore,it extensively applied as a foliar treatment on fruits,vegetables,ornamentals and turf crops from a variety of fungal diseases.In addition,it is used as an animal repellent to protect fruittrees and ornamentals from damage by rabbits,rodents,and deer.Thiram is also used in rubber industry,in the treatment of human scabies,as a sunscreen,and as a bacte-ricide in soap [5–7].Although the usability of this dithiocarbamate compound is unassailable,its increasing application results in1386-1425/$-see front matter Ó2013Elsevier B.V.All rights reserved./10.1016/j.saa.2013.05.020Corresponding author.Tel./fax:+986113331042.E-mail addresses:rastegarz@ ,rastegarz@scu.ac.ir (S.Rastegarzadeh).being released into the environment leading to contamination of water,soil,and food.These contaminants adversely affect the envi-ronment and have serious hazardous effect in the living organisms.The reported studies on rats showed that the exposure to thiram has harmful effects on hepatic system and skin such as skin lesions (e.g.hand eczema or dermatitis),hepatic dysfunctions,neurotox-icity and citotoxicity [8].The residual amounts of thiram in human diet in combination with nitrite represent the potential precursor for the formation of carcinogenic nitrosamine.Furthermore,it has been mentioned that in body,carbon disulfide is formed from the breakdown of thiram and contributes to the toxicity of thiram to the liver [9].According to the US EPA (Environmental Protection Agency),thiram is expected to be sufficiently mobile and persistent in some cases to reach surface waters in concentrations high enough to impact aquatic life [10].To evaluate the risks of its intensive use,the determination of thiram in environmental matrixes is necessary to assay the thiram residues.Various analytical methods have been reported for the determi-nation of thiram in different crop matrices and environmental samples,including mass spectrometry [3,5],high-performance liquid chromatography [10–12],spectrophotometry [7,13,14],gas chromatography [15],polarography and voltammetry [16–18]chemiluminescence analysis [6,8,19]and surface-enhanced Raman scattering spectroscopy [1].The sample preconcentration step which has attracted much attention in analytical procedure includes the extraction of inter-ested components from a sample matrix.The most popular pre-treatment methods are liquid–liquid extraction (LLE)and solid phase extraction (SPE).Nevertheless,these conventional extraction methods are laborious,time-consuming and require large volumes of samples and toxic organic solvents.In the past years,several techniques particularly microextraction have been developed for sample preconcentration,due to the many advantages which makes it more attractive with respect to classic extraction approaches.Dispersive liquid–liquid microextraction (DLLME)as a considerable microextraction technique was emerged by Assadi et al.in 2006[20],which was based on a ternary solvent system like homogeneous LLE and cloud point extraction.In this method,an appropriate mixture of extraction and dispersive solvents is in-jected rapidly into an aqueous sample,resulting in the formation of a cloudy solution.The contact area between the extracting solvent and the sample solution is extremely large;thus,the extraction equilibrium is obtained rapidly.The advantages of DLLME are the usage of a small volume of organic solvents,simplicity of the oper-ation,rapidity,low cost,high recovery,high enrichment factor and environmentally friendly nature.Recently,this method has been applied for the determination of trace organic compounds and metal ions in environmental samples [21–27].In this work,a DLLME method followed by UV–vis spectropho-tometry equipped with a microcell is applied to sensitivedetermination of thiram.A colored organic phase is formed by reaction between thiram,ethanolic KOH and copper (II)sulfate and injection of CCl 4as extractant solvent.The important parame-ters,such as reaction conditions,the type and volume of extraction solvent and extraction time are investigated and optimized.Experimental ApparatusRecording the spectra and the absorbance measurements were made by a Jenway UV–vis spectrophotometer model 6320using quartz microcells with capacity of 350l L.A Metrohm 632(Swit-zerland)pH-meter was used to measure pH with a combined glass electrode.A model BHG HERMLE centrifuge (Germany)was used for the phase separation.ReagentsAll chemicals used were of analytical grade and double distilled water was used throughout.A stock solution of 100l g mL À1of thiram was prepared by dis-solving 0.0100g of pure thiram (Merck)in ethanol and diluting to 100mL in a volumetric flask.Working standard solutions were obtained daily by successive dilutions of this stock solution.A 0.10mol L À1ethanolic KOH solution (Merck)was prepared by dis-solving 0.561g of KOH in ethanol and diluting to 100mL.A stock solution of copper (II),1000l g mL À1,was prepared by dissolving of 0.391g CuSO 4Á5H 2O (Merck)in water and diluting to 100mL.Copper (II)working standard solutions were prepared daily by stepwise dilution of the stock solution.Dispersive liquid–liquid microextraction procedureFor DLLME under optimum conditions,an aliquot of the solu-tion containing thiram was placed in a 10mL volumetric flask,then 1mL of ethanolic potassium hydroxide solution (0.1mol L À1)and 5mL of 1l g mL À1of copper (II)were added.The resulting solution was then diluted to the mark with water and mixed thoroughly.After 10min the solution was transferred to a glass test tube with a conical bottom.Since ethanol (as disperser solvent)exists in solution,only 200l L of carbon tetrachloride (as extraction sol-vent)was rapidly injected into the sample solution by a microsy-ringe.After shaking manually,a cloudy solution (water,ethanol and CCl 4)was rapidly produced,and then the mixture was centri-fuged for 5min at 5000rpm.Accordingly,the dispersed fine drop-lets of the extraction phase deposited at the bottom of the conical test tube (170±5l L).The remained organic phase was removed with a microsyringe and subsequently placed into the quartz microcell and the absorbance was measured at 430nm against the blank.A blank solution was also run under the same procedure without adding any thiram.Preparation of plant seeds samplesAppropriate amounts of tomato,cucumber and watermelon seeds were weighed and placed into a 100mL beaker,then 30mL of ethanol was added,covered by a lid and stirred for 24h.The solution was then filtered and diluted to 50mL in a vol-umetric flask.An aliquot of the solutions was treated under the recommended procedure for DLLME and subsequent determina-tion of thiramcontent.Fig.1.Chemical structure of thiram (tetramethylthiuram disulfide).and Biomolecular Spectroscopy 114(2013)46–5047Results and discussionA dispersive liquid–liquid microextraction procedure based on the reaction of thiram in ethanolic KOH solution and in the pres-ence of copper(II)sulfate was developed for preconcentration of thiram.In order tofind the appropriate conditions for DLLME,dif-ferent experimental parameters were studied and optimized using a thiram standard solution.Wavelength selectionIn order to perform quantitative analysis spectrophotometri-cally the maximum absorption wavelength should be established. Therefore,the sample solution containing different concentrations of thiram was examined according to the recommended procedure of DLLME and corresponding spectra of sedimented phase were recorded in the range of350–650nm.As seen in Fig.2,upon increasing thiram concentration the absorbance at maximum wavelength,430nm,was increased.Therefore this wavelength was selected for measuring the absorbance of the extracted phase throughout this study.Effect of ethanolic KOH solutionThe presence of ethanolic KOH has strong effect on developing the color product.For this purpose the influence of ethanolic KOH solution in the concentration range of0–0.05mol LÀ1was studied in DLLME procedure.As can be seen from Fig.S1in the absence of KOH the absorbance of sedimented phase is very low which indi-cates that the colored product has not been formed efficiency. However,the maximum absorbance due to high extraction effi-ciency was obtained at0.01mol LÀ1.Therefore,1mL of ethanolic KOH solution0.1mol LÀ1was added to10mL sample solution to achieve this concentration.The effect of volume of ethanolic KOH 0.1mol LÀ1was also studied in the range of0.2–4.0mL.No signif-icant effect was observed in volume range of0.5–2.0mL.Nature of the extraction solvent and disperser solventSince the ethanol(as a dispersive solvent)was provided by the addition of ethanolic KOH,the cloudy solution appeared with rapid injection of only the extraction solvent.Thus,the effect of type of extraction solvent on DLLME was studied.The characteristics of extraction solvent in DLLME are similar to conventional LLE,namely low solubility in water and high effi-ciency for extraction of the target analyte.Therefore,carbon tetra-chloride,chloroform,dichloromethane and cyclohexane were considered for this purpose.A cloudy solution and two-phase sys-tem was formed using these solvents,however in the case of car-bon tetrachloride the signal was higher and more reproducible. Therefore,carbon tetrachloride was selected as extraction solvent.Additional experiments showed that the injection of a mixture of CCl4and different disperser solvents including methanol,etha-nol,acetonitrile and acetone at different ratios decreased the extraction efficiency.As mentioned previously the disperser sol-vent(ethanol)exists in solution and further addition of this solvent caused part of carbon tetrachloride to be dissolved in disperser sol-vent and migrated into aqueous phase.For this reason,the dis-solved carbon tetrachloride could not be sedimented down and accordingly extraction efficiency was decreased.Finally,the sug-gested method was carried out only by injecting the extraction sol-vent and the solution was shaking manually after injection of carbon tetrachloride.Thereby,a cloudy solution was formed and the extraction occurred.Effect of extraction solvent volumeThe volume of carbon tetrachloride as extraction solvent has strong effect on the sensitivity due to its effect on enrichment fac-tor.For this purpose a series of experiments were carried out with different volumes of carbon tetrachloride in the range of200–1000l L.As it is observed in Fig.S2,the absorbance of organic phase was decreased by increasing of the CCl4volume.This could be due to the dilution effect which decreases in concentration of the extracted species in sedimented ing injection vol-umes less than200l L of CCl4lower volume of organic phase was obtained,so that the absorption signal could not be measured by spectrophotometerfitted with microcells.Thus,in order to achieve high enrichment factor and low detection limit value, 200l L of CCl4was selected as optimum extractant solvent volume throughout the experiments.Effect of copper(II)amountIn order to achieve highest extraction efficiency the effect of the copper(II)concentration in the range of0.1–0.8l g mLÀ1was investigated.The obtained results revealed that the absorbance increased with increasing of the copper(II)concentration up to 0.5l g mLÀ1and remained nearly constant above this value.There-fore,the concentration of0.5l g mLÀ1was chosen as the optimum amount of the copper(II)concentration.Effect of standing timeIt was found that the incubation time before injection of carbon tetrachloride has an effect on the formation of the colored product. Therefore the dependence of absorbance of sedimented phase upon time in the range of2–40min was studied under previously optimized conditions.The obtained results show a considerable increase in the analytical signal with incubation time up to 10min and remained constant above that.Thus the injection of extraction solvent was carried out10min after mixing of the reagents.Since the extraction solvent was injected to the solution and it appeared cloudy without the addition of disperser,for breaking up of organic phase intofine droplets[28–30],it was necessary to shake the solution after injection of extraction solvent.48S.Rastegarzadeh et al./Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy114(2013)46–50Accordingly,the effect of shaking time on the DLLME of thiram was investigated in the range of0–60s.The obtained results denoted that the highest extraction efficiency was achieved about10s and above that shaking time had no significant effect on the absor-bance of sedimented phase.As the cloudy solution appeared with-infirst few seconds the infinitely large surface area between extraction solvent and aqueous phase was achieved and the color product diffuses quickly into the extraction solvent.This is the remarkable advantage of DLLME technique.The influence of centrifugation time on DLLME of thiram was also studied.The results indicated that for a complete separation of organic and aqueous phase the mixture should be centrifuged for5min at5000rpm.Interference studiesThe effect of potential interference of cations and anions on the DLLME and determination of thiram using the proposed procedure was studied.An error of±5%in the absorbance reading was consid-ered tolerable.In these experiments,sample solutions containing of200ng mLÀ1of the thiram and different concentrations of other ions were treated under the recommended procedure using opti-mum conditions.The results given in Table S1indicate that themethod is more selective to thiram than a large number of cations and anions.Analyticalfigures of meritAfter optimization of all parameters,quantitative characteris-tics of the proposed method were studied.The linear dynamic range,correlation coefficient(r),repeatability,limit of detection (LOD)and preconcentration factor were determined to evaluate the method performance.The analytical characteristics of the opti-mized method summarized in Table1.The preconcentration factor for the suggested DLLME method is calculated by the ratio of the highest sample volume(10mL)and thefinal lowest volume (170l L).Thus,a preconcentration factor of about58.8was achieved using this procedure.Application to real samplesTo demonstrate the performance and validation of the present method,it was utilized to determine the thiram in two water samples,which were selected from Karun River(Khuzestan Prov-ince,Iran)and Tap water(Ahvaz,Iran).The tested water samples were found to be free from thiram according to the optimized pro-cedure.The recovery tests were performed by the analysis of the samples spiked with known amounts of thiram.The analytical data of this investigation are given in Table2,as can be seen,the thiram recovery for the spiked samples is quantitative(94.7–104.9%).The proposed method was then applied for the determination thiram in three plant seed samples preserved by thiram.In addi-tion for evaluation of the accuracy of the method,a comparison between results obtained by suggested method and HPLC[11] was performed.The obtained results from both methods were sta-tistically evaluated by performing Student’s t-test and F-test.As can be seen in Table3the values calculated were found to be less than tabulated values at95%confidence level indicating no signif-icant differences in the accuracy and precision of the recom-mended method and the HPLC.ConclusionsIn the present study a DLLME method has been employed for sensitive determination of thiram.Miniaturization of toxic organic solvent using dispersive liquid–liquid microextraction combined with microvolume UV–vis spectrophotometry allows the develop-ment of a green method which is environment-friendly.Besides simplicity of operation,rapidity,low sample volume,low cost and high preconcentration factor are some advantages of the sug-gested method.There is no need for additional dispersive solvent because ethanol provided by the addition of ethanolic KOH acts as a disperser too.A comparison between presented approach and previously reported method for the determination of thiramTable1Analytical characteristics of the presented DLLME method for determination of thiram.Parameter Analytical featuresLinear range(ng mLÀ1)(n=10)25–1000Correlation coefficient(r)0.9985Detection limit(ng mLÀ1)(3r,n=10)11.5Precision(RSD%for100and500ng mLÀ1,n=8) 2.7,1.1Preconcentration factor58.8Table2Determination of thiram in water samples by proposed method.Sample Added(ng mLÀ1)Found a(ng mLÀ1)Recovery(%)River water(Karun)0N.D b–250257.8±3.2103.1500524.4±5.9104.9Tap water(Ahvaz)0N.D–250241.1±2.796.4500473.6±5.394.7a Mean±standard deviation(n=5).b Not detected.Table3Determination of thiram in plant seed samples by proposed method.Seed sample Thiram found(mg gÀ1)a t-Test b F-test cProposed method HPLCTomato0.428±0.0130.451±0.016 1.93 1.51 Cucumber 1.114±0.033 1.152±0.040 1.27 1.47 Watermelon0.748±0.0210.726±0.025 1.17 1.42a Mean±standard deviation(n=3).b Tabulated t-value for four degrees of freedom at95%confidence level is2.78.c Tabulated F-value for(2,2)degrees of freedom at95%confidence level is39.Table4Comparison of the proposed method with other methods for determination of thiram.DetectiontechniqueLinear range(ng mLÀ1)LOD(ng mLÀ1)RSD(%)Ref.SERS a 3.3–400.0 2.0NG b[1]FI-CL c50.0–1000 5.0 2.6[6]Spectrophotometry0–24,000300 1.9[7]CL-ELISA d9.0–15009.0NG[8]HPLC–UV500–450088<5[10]HPLC–UV 5.0–600 1.0NG[11]Spectrophotometry500–2500330NG[13]Voltammetry240.4–144,258103.4 1.6[17]FI-CL7.5–25007.0 2.5[19]Voltammetry48–2400132–5.8[31]Spectrophotometry Up to20,000161NG[32]DLLME-UV–vis25–100011.5 1.1–2.7Thisworka Surface-Enhanced Raman Scattering.b Not given.c Flow Injection-Chemiluminescence.d Chemiluminescence-Enzyme-Linked Immuno-Sorbent Assay.S.Rastegarzadeh et al./Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy114(2013)46–5049is given in Table4.As can be seen in table the LOD of suggested method is better or comparable to many of the reported tech-niques.Furthermore the application of spectrophotometric detec-tion has merits of simplicity,cheapness and portability.This methodology gives good accuracy,low limits of detection and excellent precision which show its potentiality in analysis of thi-ram in environmental and agricultural samples.AcknowledgementThe authors are grateful to Shahid Chamran University Research Council forfinancial support of this work(Grant1391). Appendix A.Supplementary materialSupplementary data associated with this article can be found,in the online version,at /10.1016/j.saa.2013.05.020. 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